U.S. patent application number 13/198260 was filed with the patent office on 2012-02-09 for digital immunochromatographic test strip for semi-quantitative detection of aflatoxin b1 and preparation method thereof.
This patent application is currently assigned to Oil Crops Research Institute, Chinese Academy of Agricultural Science. Invention is credited to Xiaomei Chen, Xiaoxia Ding, Jun Jiang, Peiwu LI, Daohong Zhang, Qi Zhang, Wen Zhang.
Application Number | 20120034711 13/198260 |
Document ID | / |
Family ID | 43226436 |
Filed Date | 2012-02-09 |
United States Patent
Application |
20120034711 |
Kind Code |
A1 |
LI; Peiwu ; et al. |
February 9, 2012 |
Digital immunochromatographic test strip for semi-quantitative
detection of aflatoxin B1 and preparation method thereof
Abstract
The present invention belongs to the field of biological
detection. Multi-line immunochromatographic test strip for
semi-quantitative detection of aflatoxin B.sub.1 comprises a
paperboard, wherein a water-absorbing pad, a detection pad, a
gold-labeled pad and a sample pad are adhered sequentially on one
surface of the paperboard from top to bottom, wherein each adjacent
pads is overlapped and connected, the detection pad uses a
nitrocellulose film as a backing pad, the nitrocellulose film is
provided with a transverse control line, a test line I, a test line
II and a test line III, wherein the control line is coated with a
rabbit anti-mouse polyclonal antibody, and the test line I, test
line II and test line III are coated with aflatoxin B.sub.1-bovine
serum albumin conjugate (AFB.sub.1-BSA), respectively: and the
gold-labeled pad is transversely coated with a nanogold-labeled
anti-aflatoxin B.sub.1 monoclonal antibody. Said test strip is used
for semi-quantitative detection of aflatoxin B.sub.1, and is
characterized by quick detection, simple procedure and high
sensitivity.
Inventors: |
LI; Peiwu; (Wuhan, CN)
; Zhang; Daohong; (Wuhan, CN) ; Zhang; Qi;
(Wuhan, CN) ; Zhang; Wen; (Wuhan, CN) ;
Ding; Xiaoxia; (Wuhan, CN) ; Jiang; Jun;
(Wuhan, CN) ; Chen; Xiaomei; (Wuhan, CN) |
Assignee: |
Oil Crops Research Institute,
Chinese Academy of Agricultural Science
Wuhan
CN
|
Family ID: |
43226436 |
Appl. No.: |
13/198260 |
Filed: |
August 4, 2011 |
Current U.S.
Class: |
436/501 ;
156/182; 422/424 |
Current CPC
Class: |
G01N 2333/38 20130101;
G01N 33/56961 20130101 |
Class at
Publication: |
436/501 ;
422/424; 156/182 |
International
Class: |
G01N 33/566 20060101
G01N033/566; B32B 38/08 20060101 B32B038/08; G01N 31/22 20060101
G01N031/22 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 5, 2010 |
CN |
201010245094.0 |
Claims
1. A test strip for digital detection of aflatoxin B.sub.1
comprising: a backing; a sample pad, a marker-labeled pad coated
with a marker-labeled antibody against aflatoxin B.sub.1; a
detection pad comprising a control line and a plurality of test
lines, wherein the control line is coated with a secondary antibody
against said marker-labeled antibody provided in said
marker-labeled pad, and wherein said plurality of test lines are
each coated with a different concentration of aflatoxin
B.sub.1-bovine serum albumin conjugate; and a water-absorbing pad;
wherein all four pads are attached to one side of said backing with
said water-absorbing pad on top, followed by said detection pad,
said gold-labeled pad and said sample pad sequentially with said
sample pad at the bottom, each pad in direct contact with its
neighboring pad or pads.
2. The test strip of claim 1, wherein said backing comprises a
paperboard: said marker-labeled pad comprises a gold-labeled pad
transversely coated with a nanogold-labeled anti-aflatoxin B.sub.1
monoclonal antibody; and said detection pad further comprises a
nitrocellulose film on which said control line and said plurality
of test lines are disposed in a direction perpendicular to a
longitudinal axis of said test strip, and said plurality of test
lines comprise a first test line (test line I), a second test line
(test line II) and a third test line (test line III), test line I
being the closest to said control line.
3. The test strip of claim 2, wherein the distances between the
test line I, test line II and test line III on the detection pad
and the upper border of the nitrocellulose film are 11.about.17 mm,
13.about.19 mm and 15.about.21 mm, respectively, and the distances
between each two adjacent test lines are at least 2 mm; and the
distance between the control line and the test line I is 5.about.11
mm.
4. The test strip of claim 2, wherein the coating amount of
aflatoxin B.sub.1-bovine serum albumin conjugate required per cm of
the test line I, test line II and test line III on the detection
pad are 120.about.600 ng, 40.about.200 ng, and 20.about.100 ng,
respectively.
5. The test strip of claim 2, wherein the particle diameter of the
nanogold used in said gold-labeled pad is 15.about.20 nm: and the
amount of nanogold-labeled anti-aflatoxin B.sub.1 monoclonal
antibody required per cm of coating length on the gold-labeled pad
is 60.about.216 ng.
6. The test strip of claim 1, wherein said marker-labeled antibody
against aflatoxin B.sub.1 provided in said marker-labeled pad is
derived from mouse and said secondary antibody is a rabbit
anti-mouse polyclonal antibody.
7. The test strip of claim 6, wherein the coating amount of rabbit
anti-mouse polyclonal antibody required per cm of the control line
is 200.about.500 ng.
8. The test strip of claim 1, wherein the water-absorbing pad has
the length of 16.about.18 mm and the width of 2.about.4 mm; the
detection pad has the length of 25.about.30 mm and the width of
2.about.4 mm; the marker-labeled pad has the length of 6.about.9 mm
and the width of 2.about.4 mm; the sample pad has the length of
12.about.18 mm and the width of 2.about.4 mm, and overlapped parts
of each adjacent pad have the length of 1.about.3 mm.
9. A method for preparation of a test strip for aflatoxin B.sub.1
comprises the following steps: (1) preparation of a water-absorbing
pad: providing the water-absorbing pad by cutting a water-absorbing
paper; (2) preparation of a detection pad, comprising the following
steps: (a) coating a plurality of test lines: preparing
0.1.about.0.5 mg mL.sup.-1 of coating solution A using aflatoxin
B.sub.1-bovine serum albumin conjugate; and coating a
nitrocellulose film transversely with the coating solution A at
about 11.about.17 mm, 13.about.19 mm and 15.about.21 mm from the
upper border of said film by spot-spraying, resulting in a test
line I, test line II and test line III, the distances between each
test line being at least 2 mm, wherein the coating amount of
aflatoxin B.sub.1-bovine serum albumin conjugate (AFB.sub.1-BSA)
required on per cm of the test line I, test line II and test line
III is 120.about.600 ng, 40.about.200 ng, and 20.about.100 ng,
respectively, and then drying said film for 8.about.20 minutes at
37.about.40.degree. C.; (b) coating a control line: preparing
0.4.about.0.6 mg mL.sup.-1 of coating solution B using a rabbit
anti-mouse polyclonal antibody; and coating the nitrocellulose film
transversely with coating solution B 5.about.11 mm from the test
line I on the nitrocellulose film by spot-spraying to obtain the
control line, wherein the coating amount of rabbit anti-mouse
polyclonal antibody required on per cm of the control line is
200.about.500 ng, and then drying said film for 8.about.20 minutes
at 37.about.40.degree. C.; (3) preparation of a sample pad: soaking
a fiberglass film is put into a blocking solution A, then taking
out of the solution and drying for 10.about.16 hours at
37.about.40.degree. C. to obtain the sample pad, and placing in a
desiccator to store at room temperature; (4) preparation of a
gold-labeled pad: spraying a nanogold-labeled anti-aflatoxin
B.sub.1 monoclonal antibody solution transversely onto the sample
pad by spot-spraying, wherein the amount of nanogold-labeled
anti-aflatoxin B.sub.1 monoclonal antibody required per cm of
spraying length is 60.about.216 ng, then subjecting the coated pad
to lyophilization under vacuum for 2.about.6 hours, and placing in
a desiccator to store at room temperature; and (5) assembly of the
test strip for semi-quantitative detection of aflatoxin B.sub.1
attaching said water-absorbing pad, said detection pad, said
gold-labeled pad and said sample pad sequentially on one side of a
paperboard from top to bottom, such that each pad overlaps with
adjacent pad or pads, the axial length of each overlapped part is
1.about.3 mm.
10. The method of claim 9, wherein the coating solution A comprises
10.about.50 mg of commercially available aflatoxin B.sub.1-bovine
serum albumin conjugate (AFB.sub.1-BSA), 1.about.2 g bovine serum
albumin, 1.about.2 g sucrose, 0.02.about.0.05 g sodium azide, 0.8 g
sodium chloride, 0.29 g disodium hydrogen phosphate dodecahydrate,
0.02 g potassium chloride, 0.02 g potassium dihydrogen phosphate,
to which water is added to reach 100 mL of final volume; and the
coating solution B comprises 50 mg rabbit anti-mouse polyclonal
antibody, 0.02.about.0.05 g sodium azide, 0.8 g sodium chloride,
0.29 g disodium hydrogen phosphate dodecahydrate, 0.02 g potassium
chloride, 0.02 g potassium dihydrogen phosphate, to which water is
added to reach 100 mL of final volume.
11. The method of claim 9, wherein the blocking solution A
comprises 1.about.2 g bovine serum albumin, 0.1.about.0.2 mL Triton
X-100, 0.3 g polyvinylpyrrolidone, 2.about.5 g sucrose,
0.02.about.0.05 g sodium azide, 0.8 g sodium chloride, 0.29 g
disodium hydrogen phosphate dodecahydrate, 0.02 g potassium
chloride, 0.02 g potassium dihydrogen phosphate, to which water is
added to reach 100 mL of final volume.
12. The method of claim 9, wherein the method for preparing said
nanogold-labeled anti-aflatoxin B.sub.1 monoclonal antibody
solution comprises: providing 50.0 mL of commercially available
nanogold solution with the mass concentration of 0.01%, adjusting
the pH of the solution to 5.5; adding slowly with stirring 2 mL of
0.1 mg mL.sup.-1 anti-aflatoxin B.sub.1 monoclonal antibody aqueous
solution and stirring the solution for 30 min; adding 10% (w/w)
bovine serum albumin aqueous solution until the final concentration
of bovine serum albumin is 1% (w/w), and further stirring the for
30 min; after standing at 4.degree. C. for 2 hours, centrifuging at
1500 rpm for 15 min, removing supernatant and discarding the
pellet; and centrifuging the supernatant obtained at 12000 rpm for
30 min; after the supernatant is discarded, adding 50 mL
label-washing preservation solution added and centrifuging the
resulting solution at 12000 rpm for 30 min again; after the
supernatant is discarded, resuspending the precipitate obtained
with label-washing preservation solution to obtain the concentrated
solution with the volume of 5 mL, which is stored at 4.degree. C.,
wherein the mass concentration of the nanogold-labeled
anti-aflatoxin B.sub.1 monoclonal antibody solution is 0.04 mg
mL.sup.-1; and preparing said label-washing preservation solution
by mixing 2.0 g PEG-20000, 0.2 g sodium azide and 0.1235 g boric
acid, to which water is added to 1000 mL, followed by filtration
through 0.22 .mu.m filter membrane.
13. Use of the test strip of claim 1 for semi-quantitative
detection of aflatoxin B.sub.1, wherein the test sample which has
been grinded finely is weighed, to which methanol aqueous solution
with the concentration of 60.about.80% (v/v) is added, and the
rrilv ratio of the test sample and methanol solution is 2 g
mL.sup.-1; the solution is well-mixed and extracted with sonication
in 50.about.60' C. water-bath for 5.about.10 min, then left for
5.about.10 min, the supernatant (i.e. the extract) is diluted to
1:2.5 with water, resulting in that the final concentration of
methanol in the dilution is 24.about.32%; 100 .mu.L diluted sample
solution used as the test solution is added dropwise to the sample
pad of the multi-test-line digital immunochromatographic test strip
for semi-quantitative detection of aflatoxin B.sub.1, and said test
strip is used as the test strip for detection; in the meantime, 100
.mu.L of water is used as the negative control solution and added
dropwise to the sample pad of another multi-test-line digital
immunochromatographic test strip for semi-quantitative detection of
aflatoxin B.sub.1, which is used as the control test strip; and the
results are read out after 15 min; and wherein result evaluation
comprises: (1) positive: the control line of the test strip for
detection of test sample shows a red line, and if the color of test
line I among the three test lines is slightly lighter than that of
the control test strip, and those of test line II and test line III
are substantially the same as the control test strip, the content
of aflatoxin B.sub.1 in the sample is between 0.625 and 1.25 ng
g.sup.-1; if the test line I among the three test lines does not
show red, and the colors of test line II and test line III are
substantially the same as the control test strip, the content of
aflatoxin B.sub.1 in the sample is 1.25 ng g.sup.-1; if the test
line I among the three test lines does not show red, the color of
test line II is lighter than that of the control test strip, and
the color of test line III is substantially the same as the control
test strip, the content of aflatoxin B.sub.1 in the sample is
between 1.25 and 2.5 ng g.sup.-1; if the test line I and II of the
three test lines do not show red, and the color of test line III is
substantially the same as the control test strip, the content of
aflatoxin B.sub.1 in the sample is 2.5 ng g.sup.-1; if the test
line I and II of the three test lines do not show red, and the
color of test line III is lighter than that of the control test
strip, the content of aflatoxin B.sub.1 in the sample is between
2.5 and 10 ng g.sup.-1; if all the three test lines do not show
red, the content of aflatoxin B.sub.1 in the sample is not less
than 10 ng g.sup.-1; (2) negative: the control line of the test
strip for detection of test sample shows a red line, and the colors
of the three test lines are close to those of the control test
strip, then it is a negative result, which indicates the content of
aflatoxin B.sub.1 in the sample is less than 0.625 ng g.sup.-1; (3)
null: no matter the test lines of the test strip for detection of
test sample show red lines or not, the test strip is considered as
null as long as the control line does not exhibit a red line.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to and the benefit of
co-pending Chinese patent application Serial No. 201010245094.0,
filed Aug. 5, 2010, which application is incorporated herein by
reference in its entirety.
FIELD OF TECHNOLOGY
[0002] The present invention belongs to the field of biological
detection, and especially relates to a multi-test-line digital
immunochromatographic test strip for semi-quantitative detection of
aflatoxin B.sub.1 and preparation method thereof.
BACKGROUND ART
[0003] Aflatoxins are secondary metabolites mainly secreted by
Aspergillus flavus and Aspergillus parasiticus, and are a category
of natural toxic compounds which can cause serious damages to human
and animals. Among the aflatoxins which have been found, aflatoxin
B.sub.1 (referred to AFB.sub.1) is the most toxic aflatoxin, and
its toxicity, carcinogenicity and frequency of contamination are
the most severe among biotoxins.
[0004] After contaminating food and animal feed, aflatoxin will
directly or indirectly enter the human food chain, and bring risk
of health and safety for human, and its danger is proportional to
the amount of intake of aflatoxin. Aflatoxin B.sub.1 is found
widely in rice, maize, peanut, sesame, soy bean, rapeseed and other
agricultural products and other foods such as peanut butter, thus
almost all the countries in the world have defined the maximum
allowable concentration of aflatoxin B.sub.1 in food and animal
feed, and stipulated it as a mandatory standard. Therefore, it is
important for ensuring safety of food consumption to improve the
detection of aflatoxins, especially aflatoxin B.sub.1, in
particular the quick-detection to know and obtain the health
information of food and animal feed rapidly.
[0005] The conventional aflatoxin B.sub.1 detection techniques in
the prior art mainly include thin layer chromatography, precision
instruments analysis and immunoassay. The immunoassay method
established in recent years overcomes the disadvantages of the
first two methods, has advantages such as high specificity, good
sensitivity, simplified pre-treatment of sample, low cost, less
damage to laboratory personnel and much less environmental
pollution, and is suitable for testing in batches on sites. Among
the above immunological methods, the immunochromatographic
quick-detection technique based on nano-gold is simple, rapid and
sensitive, and is suitable for field testing, thus is greatly
valuable and has broad prospects for application. However,
traditional aflatoxin B.sub.1 immunochromatographic test strip only
has one test line, and can only be used for qualitative detection
of aflatoxin B.sub.1 in samples, moreover, its sensitivity is low.
Therefore, there are still needs to provide a multi-test-line
immunochromatographic test strip for aflatoxin B.sub.1 to achieve
the semi-quantitative detection of aflatoxin B.sub.1 at high,
moderate or low concentration in a sample, and it is of great
significance and high value for monitoring aflatoxin B.sub.1 in
food and agricultural products.
[0006] Usually, a traditional immunochromatographic test strip has
only one test line for one analyte and only shows whether a given
sample has the analyte at a concentration more or less than the
"threshold concentration" represented by the sole test line. While
some traditional test strips have multi-test lines, each test line
represents a threshold concentration for a different analyte. Here
we describe a new immunochromatographic test strip with multi-test
lines for the same analyte, and each of the test lines represents a
different threshold concentration. Considering its
semi-quantitative or quantitative detection feature, the
multi-test-line immunochromatographic test strip of the present
invention is also sometimes named "digital" immunochromatographic
test strip herein.
SUMMARY OF INVENTION
[0007] The technical problem to be solved by the present invention
is to provide a multi-test-line digital immunochromatographic test
strip for semi-quantitative detection of an analyte, e.g., a
biotoxin such as aflatoxin B.sub.1 and preparation methods thereof.
The multi-test-line digital immunochromatographic test strip for
semi-quantitative detection of aflatoxin is used to
semi-quantitatively detect aflatoxin B.sub.1, and has
characteristic of quick detection, with a simple procedure and high
sensitivity.
[0008] As used herein, a multi-test-line digital
immunochromatographic test strip is defined as an
immunochromatographic test strip with three or more test lines,
each of them standing for a specific and different concentration of
an analyte (e.g., aflatoxin B.sub.1), and can he used to test at
least 4 different concentrations of the analyte on the same strip.
Here the term "digital" is used to indicate the ability of the
present invention to display the value of the analyte concentration
in a relatively narrow range, thereby akin to a reading of an
actual value. In various embodiments, the range is set to satisfy
how precise the reading needs to be--the narrower the range, the
more precise the reading. This quantitative reading is made
possible because there are three or more test lines on the
immunochromatographic test strip disclosed herein and each of them
represents a different threshold concentration, which means a user
can deduce a concentration figure on such a digital
immunochromatographic test strip.
[0009] To solve said technical problems encountered in the prior
art, the present invention provides embodiments as following:
[0010] Multi-test-line digital immunochromatographic test strip for
semi-quantitative detection of an analyte, e.g., aflatoxin B.sub.1
(see FIG. 1), comprising a backing (e.g., paperboard), wherein a
water-absorbing pad, a detection pad, a marker-labeled pad and a
sample pad are adhered or attached sequentially on one side of said
backing from top to bottom, wherein each pad is in direct contact
with its neighboring pad or pads. In one embodiment, the pads
overlap with each other and are connected.
[0011] The detection pad may use a nitrocellulose film as a hacking
pad, and the nitrocellulose film is provided with a transverse
control line, and a plurality of test lines (e.g., a test line I, a
test line II and a test line III) from top to bottom. In one
feature, the test line I, test line II and test line III are each
coated with aflatoxin B.sub.1-bovine serum albumin (AFB.sub.1-BSA)
conjugate. In one embodiment, the control line and the test lines
traverse the detection pad in a direction perpendicular to a
longitudinal axis of the test strip. The control line provides
positive control for the test strip and can be viewed as a quality
control line.
[0012] The marker-labeled pad is coated with a labeled primary
antibody against the analyte, e.g., it can be transversely coated
through spraying with a nanogold-labeled anti-aflatoxin B.sub.1
monoclonal antibody. In that case, the marker-labeled pad can be
called a gold-labeled pad. The control line in the detection pad is
coated with a secondary antibody against the primary antibody
provided in the marker-labeled pad. In an embodiment where the
primary antibody used in the marker-labeled pad is derived from
mouse, the control line in the detection pad may be coated with a
rabbit anti-mouse polyclonal antibody.
[0013] According to the embodiment mentioned above, the
water-absorbing pad has the length of 16.about.18 mm and the width
of 2.about.4 mm: the detection pad has the length of 25.about.30 mm
and the width of 2.about.4 mm; the gold-labeled pad has the length
of 6.about.9 mm and the width of 2.about.4 mm; the sample pad has
the length of 12.about.18 mm and the width of 2.about.4 mm, and
overlapped parts of each adjacent pad have the length of 1.about.3
mm.
[0014] According to the embodiment mentioned above, the
water-absorbing pad is made of a water-absorbing paper. The sample
pad is where a test sample is loaded.
[0015] According to the embodiment mentioned above, the distances
between the test line I, test line II and test line III and the
upper border of the nitrocellulose film on the detection pad are
11.about.17 mm, 13.about.19 mm and 15.about.21 mm, respectively,
and the distances between each two adjacent test lines are at least
2 mm; and the distance between the control line and the test line I
is 5.about.11 mm.
[0016] According to the embodiment mentioned above, the coating
amount of aflatoxin B.sub.1-bovine serum albumin (AFB.sub.1-BSA)
conjugate required on per cm of the test line I, test line II and
test line III on the detection pad are 1201.about.600 ng,
40.about.200 ng and 20.about.100 ng, respectively, and the coating
amount of rabbit anti-mouse polyclonal antibody required on per cm
of the control line is 200.about.500 ng.
[0017] According to the embodiment mentioned above, the particle
diameter of the nanogold used in said gold-labeled pad is
15.about.20 nm.
[0018] According to the embodiment mentioned above, the amount of
nanogold-labeled anti-aflatoxin B1 monoclonal antibody required on
per cm of spraying length on the gold-labeled pad is 60.about.216
ng.
[0019] The present invention provides a method for preparation of
the high sensitive digital immunochromatographic test strip for
semi-quantitative detection of aflatoxin B.sub.1, which
comprises:
[0020] (1) preparation of the water-absorbing pad,
[0021] wherein the water-absorbing pad is obtained by cutting a
water-absorbing paper;
[0022] (2) preparation of the detection pad, including the
following steps:
[0023] Coating of the test lines:
[0024] wherein commercially available aflatoxin B.sub.1-bovine
serum albumin conjugate (AFB.sub.1-BSA) is used to prepare
0.1.about.0.5 mg mL.sup.-1 of coating solution A, and the
nitrocellulose film is coated transversely with the coating
solution A along the positions of 11.about.17 mm, 13.about.19 mm
and 15.about.21 mm from the upper border of said film by
spot-spraying, resulting in the test line I, test line II and test
line III, the distances between each test line are at least 2 mm,
and the coating amount of aflatoxin B.sub.1-bovine serum albumin
conjugate (AFB.sub.1-BSA) required on per cm of the test line I,
test line II and test line III is 120.about.600 ng, 40.about.200
ng, and 20.about.100 ng, respectively, then said film is dried for
8.about.20 minutes at 37.about.40.degree. C.;
[0025] Coating of the control line:
[0026] wherein the rabbit anti-mouse polyclonal antibody is used to
prepare 0.4.about.0.6 mg mL-1 of coating solution B; and the
nitrocellulose film is coated transversely with coating solution B
at the position of 5.about.11 mm from the test line I on the
nitrocellulose film by spot-spraying to obtain the control line,
and the coating amount of rabbit anti-mouse polyclonal antibody
required on per cm of the control line is 200.about.500 ng, then
said film is dried for 8.about.20 minutes at 37.about.40.degree.
C.;
[0027] (3) preparation of the sample pad
[0028] wherein the fiberglass film is put into blocking solution A
to be soaked, then taken out of the solution and dried for
10.about.16 h at 37.about.40.degree. C. to obtain the sample pad,
and placed in a desiccator/drier to store at room temperature;
[0029] (4) preparation of the gold-labeled pad
[0030] wherein the nanogold-labeled anti-aflatoxin B.sub.1
monoclonal antibody solution is transversely sprayed onto the
sample pad by spot-spraying, the amount of nanogold-labeled
anti-aflatoxin B.sub.1 monoclonal antibody required on per cm of
spraying length is 60.about.216 ng, and after lyophilization under
vacuum for 2.about.6 h, the gold-labeled pad is prepared and placed
in a desiccator to store at room temperature;
[0031] (5) assembly of the multi-test-line digital
immunochromatographic test strip for semi-quantitative detection of
aflatoxin B1
[0032] wherein a water-absorbing pad, a detection pad, a
gold-labeled pad and a sample pad are adhered sequentially on one
surface of said paperboard from top to bottom, wherein each
adjacent pads are overlapped and connected, and the length of each
overlapped part is 1.about.3 mm, thus the multi-test-line digital
immunochromatographic test strip for semi-quantitative detection of
aflatoxin B.sub.1 is prepared (see FIG. 1 and FIG. 2).
[0033] According to the embodiment mentioned above, the coating
solution A comprises 10.about.50 mg of commercially available
aflatoxin B.sub.1-bovine serum albumin conjugate (AFB.sub.1-BSA),
1.about.2 g bovine serum albumin, 1.about.2 g sucrose,
0.02.about.0.05 g sodium azide, 0.8 g sodium chloride, 0.29 g
disodium hydrogen phosphate dodecahydrate, 0.02 g potassium
chloride, 0.02 g potassium dihydrogen phosphate, to which water is
added to reach 100 mL of final volume;
[0034] The coating solution B comprises 50 mg rabbit anti-mouse
polyclonal antibody, 0.02.about.0.05 g sodium azide, 0.8 g sodium
chloride, 0.29 g disodium hydrogen phosphate dodecahydrate, 0.02 g
potassium chloride, 0.02 g potassium dihydrogen phosphate, to which
water is added to reach 100 mL of final volume.
[0035] According to the embodiment mentioned above, the blocking
solution A comprises 1.about.2 g bovine serum albumin,
0.1.about.0.2 mL Triton X-100, 0.3 g polyvinylpyrrolidone,
2.about.5 g sucrose, 0.02.about.0.05 g sodium azide, 0.8 g sodium
chloride, 0.29 g disodium hydrogen phosphate dodecahydrate, 0.02 g
potassium chloride, 0.02 g potassium dihydrogen phosphate, to which
water is added to reach 100 mL of final volume.
[0036] According to the embodiment mentioned above, the method for
preparation of nanogold-labeled anti-aflatoxin B.sub.1 monoclonal
antibody solution is described as follows: 50.0 mL of commercially
available nanogold solution is taken with the mass concentration of
0.01%, and the pH of the solution is adjusted to 5.5; 2 mL of 0.1
mg mL.sup.-1 anti-aflatoxin B.sub.1 monoclonal antibody aqueous
solution is added slowly with stirring and the solution is further
stirred for 30 min; 10% (w/w) bovine serum albumin aqueous solution
is added until the final concentration of bovine serum albumin
(BSA) is 1% (w/w), and the solution is further stirred for 30 min;
after standing at 4.degree. C. for 2 h, the solution is centrifuged
at 1500 rpm for 15 min. The supernatant is removed and the pellet
is discarded: the supernatant obtained is centrifuged at 12000 rpm
for 30 min, and the supernatant is discarded, then 50 mL
label-washing preservation solution is added and the resulting
solution is centrifuged at 12000 rpm for 30 min again. The
supernatant is discarded, and the precipitate obtained is
resuspended with label-washing preservation solution to obtain the
concentrated solution with the volume of 5 mL, which is stored at
4.degree. C. wherein the mass concentration of the nanogold-labeled
anti-aflatoxin B.sub.1 monoclonal antibody solution is 0.04 mg
mL.sup.-1.
[0037] Said label-washing preservation solution is prepared by
mixing 2.0 g polyethylene glycol-20000 (PEG-20000), 0.2 g sodium
azide and 0.1235 g boric acid, to which water is added to 1000 mL,
followed by filtration through 0.22 .mu.m filter membrane.
[0038] The use of said multi-test-line digital
immunochromatographic test strip for semi-quantitative detection of
aflatoxin B.sub.1: the test sample grinded finely is weighed, to
which methanol aqueous solution with the concentration of
60.about.80% (v/v) is added, and the m/v ratio of the test sample
and methanol solution is 2 g mL.sup.-1. The solution is well-mixed
and extracted with sonication in 50.about.60.degree. C. water-bath
for 5.about.10 min, then left for 5.about.10 min. The supernatant
(i.e. the extract) is diluted to 1:2.5 with water, resulting in
that the final concentration of methanol in the dilution is
24.about.32%. 100 .mu.L diluted sample solution used as the test
solution is added dropwise to the sample pad of the multi-test-line
digital immunochromatographic test strip for semi-quantitative
detection of aflatoxin B.sub.1. Said test strip is used as the test
strip for detection. In the meantime, 100 .mu.L of water is used as
the negative control solution and added dropwise to the sample pad
of another multi-test-line digital immunochromatographic test strip
for semi-quantitative detection of aflatoxin B.sub.1, and said test
strip is used as the control test strip. The results are read out
after 15 min.
[0039] Result evaluation: (1) positive: the control line of the
test strip for detection of test sample shows a red line, if the
color of test line I among the three test lines is slightly lighter
than that of the control test strip, and those of test line II and
test line III are substantially the same as the control test strip,
the content of aflatoxin B.sub.1 in the sample is between 0.625 and
1.25 ng g.sup.-1; if the test line I among the three test lines
does not show red, and the colors of test line II and test line III
are substantially the same as the control test strip, the content
of aflatoxin B.sub.1 in the sample is 1.25 ng g.sup.-1: if the test
line I among the three test lines does not show red, the color of
test line II is lighter than that of the control test strip, and
the color of test line III is substantially the same as the control
test strip, the content of aflatoxin B.sub.1 in the sample is
between 1.25 and 2.5 ng g.sup.-1: if the test line I and II of the
three test lines do not show red, and the color of test line III is
substantially the same as the control test strip, the content of
aflatoxin B, in the sample is 2.5 ng g.sup.-1: if the test line I
and II of the three test lines do not show red, and the color of
test line III is lighter than that of the control test strip, the
content of aflatoxin B.sub.1 in the sample is between 2.5 and 10 ng
g.sup.-1; if all the three test lines do not show red, the content
of aflatoxin B.sub.1 in the sample is not less than 10 ng g.sup.-1.
(2) negative: the control line of the test strip for detection of
test sample shows a red line, and the colors of the three test
lines are close to those of the control test strip, then it is a
negative result, which indicates the content of aflatoxin B.sub.1
in the sample is less than 0.625 ng g.sup.31 1. (3) Null: no matter
the test lines of the test strip for detection of test sample show
red lines or not, the test strip is considered as null as long as
the control line does not exhibit a red line.
[0040] The working principle of the test strip is as following:
when the sample solution is added to the sample pad in the bottom
of the test strip, it moves toward the water-absorbing pad due to
capillarity, and when it moves to the gold-labeled pad, the
nanogold-labeled anti-aflatoxin B.sub.1 monoclonal antibody is
dissolved. If the sample contains aflatoxin B.sub.1, aflatoxin
B.sub.1 will bind to the nanogold-labeled anti-aflatoxin B.sub.1
monoclonal antibody on the gold-labeled pad and move upward
together, when they reach the three test lines fixed with antigen,
the antigen and aflatoxin B.sub.1 will competitively bind to the
finite antigen binding sites on the nanogold-labeled anti-aflatoxin
B.sub.1 monoclonal antibody. The more aflatoxin B.sub.1 contained
in the sample, the less nanogold-labeled anti-aflatoxin B.sub.1
monoclonal antibody the antigen on the test lines can bind, the
less colored line generates, and the lighter the color is. If the
amount of the nanogold-labeled anti-aflatoxin B.sub.1 monoclonal
antibody bound by the antigen is less than a certain value, no red
line can be exhibited by the test lines. No matter the sample
contains aflatoxin B.sub.1 or not, nanogold-labeled anti-aflatoxin
B.sub.1 monoclonal antibody which is not captured by the test lines
or the complex of the nanogold-labeled anti-aflatoxin B.sub.1
monoclonal antibody and aflatoxin B.sub.1 will move on to the
control line, and bind to the second antibody thereon, thus being
enriched and showing its color. Therefore, no matter the sample
contains aflatoxin B.sub.1 or not, the control line will be
colored. When the sample does not contain aflatoxin B.sub.1 (i.e.
negative), the strip appears four red lines, i.e. the control line
and three test lines; when the sample contains certain amount of
aflatoxin B.sub.1 (i.e. positive), six possible results may be
observed from the test strip after completion of detection: (1) the
test line I is pale red, both test line II and test line III are
red, and the control line is red; (2) the test line I does not show
red, both test line II and test line III are red, and the control
line is red; (3) the test line I does not show red, the test line
II is pale red, the test line III is red, and the control line is
red; (4) both the test line I and the test line II do not show red,
the test line III is red, and the control line is red; (5) both the
test line I and the test line II do not show red, the test line III
is pale red, and the control line is red; (6) none of the test line
I, test line II and test line III is colored, and the control line
is red; if the control line does not show red, the test strip is
null.
[0041] By application of the strip, four or more than four
concentrations of aflatoxin B.sub.1 can be detected on the same
strip.
[0042] The beneficial effects brought by the present invention
include:
[0043] (1) semi-quantitative detection of aflatoxin B.sub.1. The
multi-test-line digital immunochromatographic test strip for
semi-quantitative detection of aflatoxin B.sub.1 according to the
present invention comprises three test lines, and can be used for
semi-quantitative detection of aflatoxin B.sub.1 at high, moderate
or low concentration, which is greatly valuable for practical
application.
[0044] (2) Simplified pre-treatment of sample. To pre-treat
samples, one only needs to add methanol to the sample that has bee
grinded finely, and to extract with ultrasound technology. After
letting the extract stand to settle, the supernatant removed and
diluted for detection. Thus, the entire process of sample
pre-treatment is simple and fast.
[0045] (3) Simple operation procedure. Using the multi-test-line
digital immunochromatographic test strip for semi-quantitative
detection of aflatoxin B.sub.1, one only needs to add, drop by
drop, the extract solution from the sample to the sample pad of
test strip, which is a one-step operation and do not need any
professionals, thus the procedure is simple and convenient.
[0046] (4) Being friendly to the environment. The detection process
does not require the standard solution of aflatoxin B.sub.1 as a
positive control. The present invention provides a test strip which
does not need to add the standard solution of aflatoxin B.sub.1 as
a positive control and only needs water as a negative control when
detecting samples, thus avoiding any second-time pollution by
aflatoxin B.sub.1.
[0047] (5) High sensitivity. The lowest detectable limit of the
aflatoxin B.sub.1 in a sample using the semi-quantitative
multi-test-line digital immunochromatographic test strip of the
present invention is 0.625 ng g.sup.-1, which is lower than the
lowest detection limit of aflatoxin B.sub.1 in food regulated by
the EU.
BRIEF DESCRIPTION OF DRAWINGS
[0048] FIG. 1 is a diagram of the multi-test-line digital
immunochromatographic test strip for semi-quantitative detection of
aflatoxin B.sub.1 according to the present invention, wherein 1:
paperboard; 2: water-absorbing pad; 3: detection pad; 4:
gold-labeled pad; 5: sample pad; 6: control line; 7: test line I;
8: test line II; 9: test line III.
[0049] FIG. 2 is a side view of multi-test-line digital
immunochromatographic test strip for semi-quantitative detection of
aflatoxin B.sub.1 according to the present invention in side view,
wherein 1: paperboard; 2: water-absorbing pad; 3: detection pad; 4:
gold-labeled pad; 5: sample pad.
[0050] FIG. 3 is a representative diagram of the result of example
1, wherein 1: control test strip; 2: test strip for detection; 3:
control line; 4: test line I; 5: test line II; 6: test line
III.
[0051] FIG. 4 is a representative diagram of the result of example
2, wherein 1: control test strip; 2: test strip for detection; 3:
control line: 4: test line I; 5: test line II; 6: test line
III.
[0052] FIG. 5 is a representative diagram of the result of example
3, wherein 1: control test strip: 2: test strip for detection; 3:
control line; 4: test line I; 5: test line II; 6: test line
III.
DETAILED DESCRIPTION OF THE INVENTION
EXAMPLES 1-3
Preparation of High Sensitive Digital Immunochromatographic Test
Strip for Semi-Quantitative Detection of Aflatoxin and Use
Thereof
[0053] Commercially available anti-aflatoxin B.sub.1 monoclonal
antibody A9555 was used in the following examples 1-3. However, the
antibodies which can be used in the present invention are not
limited to the specific antibody used in the following examples,
other aflatoxin B.sub.1 antibodies are also applicable, and the
difference between them only exists in detection sensitivity.
Example 1
[0054] The method for preparation of high sensitive digital
immunochromatographic test strip for semi-quantitative detection of
aflatoxin B.sub.1 comprised the following steps:
[0055] (1) preparation of the water-absorbing pad
[0056] The water-absorbing pad was obtained by cutting a
water-absorbing paper into the size of 16 mm.times.3 mm;
[0057] (2) preparation of the detection pad
[0058] Coating of the test lines:
[0059] Commercially available aflatoxin B.sub.1-bovine serum
albumin conjugate (AFB.sub.1-BSA) was used to prepare 0.1 mg
mL.sup.-1 of coating solution A, and the nitrocellulose film was
coated transversely with coating solution A along the positions of
13 mm, 15 mm and 17 mm from the upper border of said film by
spot-spraying, resulting in the test line I, test line II and test
line III, and the coating amount of aflatoxin B.sub.1-bovine serum
albumin conjugate (AFB.sub.1-BSA) required on per cm of the test
line I, test line II and test line III was 120 ng, 40 ng and 20 ng,
respectively, then said film was dried for 8 minutes at 37.degree.
C.;
[0060] The coating solution A included 10 mg of commercially
available aflatoxin B.sub.1-bovine serum albumin conjugate
(AFB.sub.1-BSA), 2 g bovine serum albumin, 2 g sucrose, 0.02 g
sodium azide, 0.8 g sodium chloride, 0.29 g disodium hydrogen
phosphate dodecahydrate, 0.02 g potassium chloride, 0.02 g
potassium dihydrogen phosphate, to which water was added to reach
100 mL of final volume.
[0061] Coating of the control line:
[0062] The rabbit anti-mouse polyclonal antibody was used to
prepare 0.4 mg mL.sup.-1 of coating solution B; and the
nitrocellulose film was coated transversely with coating solution B
at the position of 5 mm from the test line I on the nitrocellulose
film by spot-spraying to obtain the control line, and the coating
amount of rabbit anti-mouse polyclonal antibody required on per cm
of the control line was 200 ng, then said film was dried for 8
minutes at 37.degree. C.;
[0063] The coating solution B included 50 mg rabbit anti-mouse
polyclonal antibody, 0.02 g sodium azide, 0.8 g sodium chloride,
0.29 g disodium hydrogen phosphate dodecahydrate, 0.02 g potassium
chloride, 0.02 g potassium dihydrogen phosphate, to which water was
added to reach 100 mL of final volume.
[0064] The nitrocellulose film had a length of 25 mm and a width of
3 mm.
[0065] (3) preparation of the sample pad
[0066] The fiberglass film was cut into the size of 15 mm.times.3
mm and put into blocking solution A to be soaked, then taken out of
the solution and dried for 10 h at 37.degree. C. to obtain the
sample pad, and placed in a desiccator to store at room
temperature;
[0067] The blocking solution A included 2 g bovine serum albumin;
0.1 mL Triton X-100, 0.3 g polyvinylpyrrolidone, 2.5 g sucrose,
0.02 g sodium azide, 0.8 g sodium chloride, 0.29 g disodium
hydrogen phosphate dodecahydrate, 0.02 g potassium chloride, 0.02 g
potassium dihydrogen phosphate, to which water was added to reach
100 mL of final volume.
[0068] (4) preparation of the gold-labeled pad
[0069] The sample pad was cut into the size of 8 mm.times.3 mm, and
the nanogold-labeled anti-aflatoxin B.sub.1 monoclonal antibody
solution was transversely sprayed onto the sample pad by
spot-spraying, the amount of nanogold-labeled anti-aflatoxin
B.sub.1 monoclonal antibody required on per cm of spraying length
was 192 ng. After lyophilization under vacuum for 6 h, the
gold-labeled pad was prepared and placed in a desiccator to store
at room temperature:
[0070] The method for preparation of 0.04 mg mL.sup.-1
nanogold-labeled anti-aflatoxin B.sub.1 monoclonal antibody
solution is described as follows: 50.0 mL of commercially available
nanogold solution was taken with the mass concentration of 0.01%,
and the pH of the solution was adjusted to 5.5 with 0.1 mol
L.sup.-1 potassium carbonate aqueous solution; 2 mL of 0.1 mg
mL.sup.-1 anti-aflatoxin B.sub.1 monoclonal antibody aqueous
solution was added slowly with stirring and the solution was
further stirred for 30 min; 10% (w/w) bovine serum albumin aqueous
solution was added until the final concentration of bovine serum
albumin is 1% (w/w), and the solution was further stirred for 30
min; after standing at 4.degree. C. for 2 h, it was centrifuged at
1500 rpm for 15 min. The supernatant was removed and the pellet is
discarded; the supematant obtained was centrifuged at 12000 rpm for
30 min, and the supernatant was discarded, then 50 mL label-washing
preservation solution was added and the resulting solution was
centrifuged at 12000 rpm for 30 min again. The supernatant was
discarded, and the precipitate obtained was resuspended with
label-washing preservation solution to obtain the concentrated
solution with the volume of 5.0 mL, which was stored at 4.degree.
C. for use, wherein the mass concentration of the nanogold-labeled
anti-aflatoxin B.sub.1 monoclonal antibody solution was 0.04 mg
mL.sup.-1;
[0071] The particle diameter of the nanogold in said nanogold
solution was 15 nm;
[0072] Said 0.1 mol L.sup.-1 potassium carbonate solution was
obtained as follows: 13.8 g potassium carbonate was dissolved in
purified water and diluted to 1000 mL of final volume, then
filtered by 0.22 .mu.m filter membrane; said 0.1 mg mL.sup.-1
anti-aflatoxin B.sub.1 monoclonal antibody solution was obtained as
follows: 1 mg of commercially available anti-aflatoxin B.sub.1
monoclonal antibody was diluted with water to 10 mL of final
volume; said 10% bovine serum albumin aqueous solution was obtained
as follows: 10 g bovine serum albumin was diluted with water to 100
mL, then filtered by 0.22 .mu.m filter membrane: said label-washing
preservation solution was obtained by mixing 2.0 g PEG-20000, 0.2 g
sodium azide and 0.1235 g boric acid, to which water was added to a
final volume of 1000 mL, followed by filtration through 0.22 .mu.m
filter membrane.
[0073] (5) assembly of the multi-test-line digital
immunochromatographic test strip for semi-quantitative detection of
aflatoxin B.sub.1
[0074] A water-absorbing pad, a detection pad, a gold-labeled pad
and a sample pad were adhered sequentially on one surface of said
paperboard from top to bottom, wherein each adjacent pads were
overlapped and connected, and the length of each overlapped part
was 1 mm, thus the multi-test-line digital immunochromatographic
test strip for detection of aflatoxin B.sub.1 was prepared (see
FIG. 1 and FIG. 2).
[0075] The method for use of the multi-test-line digital
immunochromatographic test strip for semi-quantitative detection of
aflatoxin B.sub.1 prepared above is described as follows: the test
samples of 1-6 which had been grinded finely were weighed, to which
methanol aqueous solution with the concentration of 60.about.80%
(v/v) was added, and the m/v ratio of the test sample and methanol
solution was 2 g mL.sup.-1. The solution was well-mixed and
extracted with ultrasound at 50.degree. C. water bath for 8 min,
then left for 10 min. The supernatant (i.e. the extract) was
diluted to 1:2.5 with water, resulting in a final concentration of
methanol in the dilution of 32%. 100 .mu.L diluted sample solution
used as the test solution was added dropwise to the sample pad of
the multi-test-line digital immunochromatographic test strip for
semi-quantitative detection of aflatoxin B.sub.1. Said test strip
was used as the test strip for detection. In the meantime, 100
.mu.L of water was used as the negative control solution and added
dropwise to the sample pad of another multitest-line digital
immunochromatographic test strip for semi-quantitative detection of
aflatoxin B.sub.1, which was used as the control test strip. The
results were read out after 15 min.
[0076] Result evaluation: the control line of the test strip for
detection of the 1st test sample showed a red line, while the color
of test line I among the three test lines was slightly lighter than
that of the control test strip, and those of test line II and test
line III were substantially the same as the control test strip,
thus it was a positive result, which indicates that the content of
aflatoxin B.sub.1 in the sample is between 0.625 and 1.25 ng
g.sup.-1, see FIG. 3-1;
[0077] The control line of the test strip for detection of the 2nd
test sample showed a red line, while the test line I among the
three test lines did not show red, and the colors of test line II
and test line III were substantially the same as the control test
strip, thus it was a positive result, which indicated that the
content of aflatoxin B.sub.1 in the sample was 1.25 ng g.sup.-1,
see FIG. 3-2:
[0078] The control line of the test strip for detection of the 3rd
test sample showed a red line, while the test line I among the
three test lines did not show red, the color of test line II was
lighter than that of the control test strip, and the color of test
line III was substantially the same as the control test strip, thus
it was a positive result, which indicated that the content of
aflatoxin B.sub.1 in the sample was between 1.25 and 2.5 ng
g.sup.-1: see FIG. 3-3;
[0079] The control line of the test strip for detection of the 4th
test sample showed a red line, while the test line I and II of the
three test lines did not show red, and the color of test line III
was substantially the same as the control test strip, thus it was a
positive result, which indicated that the content of aflatoxin
B.sub.1 in the sample was 2.5 ng g.sup.-1, see FIG. 3-4:
[0080] The control line of the test strip for detection of the 5th
test sample showed a red line, while the test line I and II of the
three test lines did not show red, and the color of test line III
was lighter than that of the control test strip, thus it was a
positive result, which indicated that the content of aflatoxin
B.sub.1 in the sample was between 2.5 and 10 ng g.sup.-1, see FIG.
3-5;
[0081] The control line of the test strip for detection of the 6th
test sample showed a red line, while all the three test lines did
not show red, thus it was a positive result, which indicated that
the content of aflatoxin B.sub.1 in the sample was over 10 ng
g.sup.-1, see FIG. 3-6.
Example 2
[0082] The method for preparation of the high sensitive digital
immunochromatographic test strip for semi-quantitative detection of
aflatoxin B.sub.1 included the following steps:
[0083] (1) preparation of the water-absorbing pad
[0084] The water-absorbing pad was obtained by cutting a
water-absorbing paper into the size of 17 mm.times.2 mm:
[0085] (2) preparation of the detection pad
[0086] Coating of the test lines:
[0087] Commercially available aflatoxin B1-bovine serum albumin
conjugate (AFB.sub.1-BSA) was used to prepare 0.25 mg mL.sup.-1 of
coating solution A, and the nitrocellulose film was coated
transversely with coating solution A along the positions of 15 mm,
17 mm, 19 mm from the upper border of said film by spot-spraying,
resulting in the test line I, test line II and test line III, and
the coating amount of aflatoxin B.sub.1-bovine serum albumin
conjugate (AFB.sub.1-BSA) required on per cm of the test line I,
test line II and test line III was 300 ng, 100 ng and 50 ng,
respectively, then said film is dried for 10 min at 38.degree.
C.;
[0088] The coating solution A comprises 25 mg of commercially
available aflatoxin B.sub.1-bovine serum albumin conjugate
(AFB.sub.1-BSA), 1 g bovine serum albumin, 2 g sucrose, 0.03 g
sodium azide, 0.8 g sodium chloride, 0.29 g disodium hydrogen
phosphate dodecahydrate, 0.02 g potassium chloride, 0.02 g
potassium dihydrogen phosphate, to which water was added to reach
100 mL of final volume.
[0089] Coating of the control line:
[0090] The rabbit anti-mouse polyclonal antibody was used to
prepare 0.5 mg mL.sup.-1 of coating solution B; and the
nitrocellulose film was coated transversely with coating solution B
at the position of 7 mm from the test line I on the nitrocellulose
film by spot-spraying to obtain the control line, and the coating
amount of rabbit anti-mouse polyclonal antibody required on per cm
of the control line was 300 ng, then said film was dried for 15 min
at 38.degree. C.;
[0091] The coating solution B included 50 mg rabbit anti-mouse
polyclonal antibody, 0.03 g sodium azide, 0.8 g sodium chloride,
0.29 g disodium hydrogen phosphate dodecahydrate, 0.02 g potassium
chloride, 0.02 g potassium dihydrogen phosphate, to which water was
added to reach 100 mL for the final volume.
[0092] The nitrocellulose film had a length of 28 mm and a width of
2 mm.
[0093] (3) preparation of the sample pad
[0094] The fiberglass film was cut into the size of 16 mm.times.2
mm and put into blocking solution A to be soaked, then taken out of
the solution and dried for 12 h at 38.degree. C. to obtain the
sample pad, and placed in a desiccator to store at room
temperature;
[0095] The blocking solution A comprises 1.about.2 g bovine serum
albumin, 0.1.about.0.2 mL Triton X-100, 0.3 g polyvinylpyrrolidone,
2.about.5 g sucrose, 0.02.about.0.05 g sodium azide, 0.8 g sodium
chloride, 0.29 g disodium hydrogen phosphate dodecahydrate, 0.02 g
potassium chloride, 0.02 g potassium dihydrogen phosphate, to which
water was added to reach 100 mL of final volume.
[0096] (4) preparation of the gold-labeled pad
[0097] The sample pad was cut into the size of 6 mm.times.2 mm, and
the nanogold-labeled anti-aflatoxin B.sub.1 monoclonal antibody
solution was transversely sprayed onto the sample pad by
spot-spraying, the amount of nanogold-labeled anti-aflatoxin
B.sub.1 monoclonal antibody required on per cm of spraying length
was 120 ng. After lyophilization under vacuum for 5 h, the
gold-labeled pad was prepared and placed in a desiccator to store
at room temperature:
[0098] The method for preparation of nanogold-labeled
anti-aflatoxin B.sub.1 monoclonal antibody solution is described as
follows: 50.0 mL of commercially available nanogold solution was
taken with the mass concentration of 0.01%, and the pH of the
solution was adjusted to 5.5 with 0.1 mol L.sup.-1 potassium
carbonate aqueous solution; 2 mL of 0.1 mg mL.sup.-1 anti-aflatoxin
B.sub.1 monoclonal antibody aqueous solution was added slowly with
stirring and the solution was further stirred for 30 min: 10% (w/w)
bovine serum albumin aqueous solution was added until the final
concentration of bovine serum albumin is 1% (w/w), and the solution
was further stirred for 30 min; after standing at 4.degree. C. for
2 h, it was centrifuged at 1500 rpm for 15 min. The supernatant was
removed and the pellet was discarded; the supernatant obtained was
centrifuged at 12000 rpm for 30 min, and the supernatant was
discarded, then 50 mL label-washing preservation solution was added
and the resulting solution was centrifuged at 12000 rpm for 30 min
again. The supernatant was discarded, and the precipitate obtained
was resuspended with label-washing preservation solution to obtain
the concentrated solution with the volume of 5.0 mL, which was
stored at 4.degree. C. for later use, wherein the mass
concentration of the nanogold-labeled anti-aflatoxin B.sub.1
monoclonal antibody solution was 0.04 mg mL.sup.-1;
[0099] The particle diameter of the nanogold in said nanogold
solution was 18 nm;
[0100] Said 0.1 mol L-1 potassium carbonate solution was obtained
as follows: 13.8 g potassium carbonate was dissolved in purified
water and diluted to 1000 mL of filial volume, then filtered by
0.22 .mu.m filter membrane; said 0.1 mg mL.sup.-1 anti-aflatoxin
B.sub.1 monoclonal antibody solution was obtained as follows: 1 mg
of commercially available anti-aflatoxin B.sub.1 monoclonal
antibody was diluted with water to 10 mL of final volume; said 10%
bovine serum albumin aqueous solution was obtained as follows: 10 g
bovine serum albumin was diluted with water to 100 mL, then
filtered by 0.22 .mu.m filter membrane; said label-washing
preservation solution was obtained by mixing 2.0 g PEG-20000, 0.2 g
sodium azide and 0.1235 g boric acid, to which water is added to
1000 mL, followed by filtration through 0.22 .mu.m filter
membrane.
[0101] (5) assembly of the multi-test-line digital
immunochromatographic test strip for semi-quantitative detection of
aflatoxin B.sub.1
[0102] A water-absorbing pad, a detection pad, a gold-labeled pad
and a sample pad were adhered sequentially on one surface of said
paperboard from top to bottom, wherein each adjacent pads were
overlapped and connected, and the length of each overlapped part
was 2 mm, thus the multi-test-line digital immunochromatographic
test strip for semi-quantitative detection of aflatoxin B.sub.1 was
prepared (see FIG. 1 and FIG. 2).
[0103] The method for use of the multi-test-line digital
immunochromatographic test strip for semi-quantitative detection of
aflatoxin B.sub.1 is described as follows: the test samples which
had been grinded finely were weighed, to which methanol aqueous
solution with the concentration of 60.about.80% (v/v) was added,
and the m/v ratio of the test sample and methanol solution was 2 g
mL.sup.-1. The solution was well-mixed and extracted with
sonication at 50.degree. C. water bath for 5 min, then left for 5
min. The supernatant (i.e. the extract) was diluted to 1:2.5 with
water, resulting in that the final concentration of methanol in the
dilution was 28%. 100 .mu.L diluted sample solution used as the
test solution was added dropwise to the sample pad of the
multi-test-line digital immunochromatographic test strip for
semi-quantitative detection of aflatoxin B.sub.1. Said test strip
was used as the test strip for detection. In the meantime, 100
.mu.L of water was used as the negative control solution and added
dropwise to the sample pad of another multi-test-line digital
immunochromatographic test strip for semi-quantitative detection of
aflatoxin B.sub.1, which was used as the control test strip. The
results were read out after 15 min.
[0104] Result evaluation: The control line of the test strip for
detection of the test sample showed a red line, while the colors of
the three test lines were close to those of the control test strip,
thus it was judged as a negative result, see FIG. 4, which
indicated the content of aflatoxin B.sub.1 in the sample was less
than 0.625 ng.sup.-.
Example 3
[0105] The method for preparation of high sensitive digital
immunochromatographic test strip for semi-quantitative detection of
aflatoxin B.sub.1 comprised the following steps:
[0106] (1) preparation of the water-absorbing pad
[0107] The water-absorbing pad was obtained by cutting a
water-absorbing paper into the size of 18 mm.times.4 mm;
[0108] (2) preparation of the detection pad
[0109] Coating of the test lines:
[0110] Commercially available aflatoxin B.sub.1-bovine serum
albumin conjugate (AFB.sub.1-BSA) was used to prepare 0.5 mg
mL.sup.-1 of coating solution A, and the nitrocellulose film was
coated transversely with coating solution A alone the positions of
17 mm, 19 mm, 21 mm from the upper border of said film by
spot-spraying, resulting in the test line I, test line II and test
line III, and the coating amount of aflatoxin B.sub.1-bovine serum
albumin conjugate (AFB.sub.1-BSA) required on per cm of the test
line I, test line II and test line III is 600 ng, 200 ng and 100
ng, respectively, then said film was dried for 10 minutes at
39.degree. C.;
[0111] The coating solution A included 50 mg of commercially
available aflatoxin B.sub.1-bovine serum albumin conjugate
(AFB.sub.1-BSA), 1.5 g bovine serum albumin, 1.5 g sucrose, 0.02 g
sodium azide, 0.8 g sodium chloride, 0.29 g disodium hydrogen
phosphate dodecahydrate, 0.02 g potassium chloride, 0.02 g
potassium dihydrogen phosphate, to which water was added to reach
100 mL of final volume.
[0112] Coating of the control line:
[0113] Rabbit anti-mouse polyclonal antibody was used to prepare
0.6 mg mL.sup.-1 of coating solution B: and the nitrocellulose film
was coated transversely with coating solution B at the position of
9 mm from the test line I on the nitrocellulose film by
spot-spraying to obtain the control line, and the coating amount of
rabbit anti-mouse polyclonal antibody required on per cm of the
control line was 500 ng, then said film was dried for 10 minutes at
39.degree. C.;
[0114] The coating solution B comprises 50 mg rabbit anti-mouse
polyclonal antibody, 0.02 g sodium azide, 0.8 g sodium chloride,
0.29 g disodium hydrogen phosphate dodecahydrate, 0.02 g potassium
chloride, 0.02 g potassium dihydrogen phosphate, to which water was
added to reach 100 mL of final volume.
[0115] The nitrocellulose film had a length of 30 mm and a width of
4 mm.
[0116] (3) preparation of the sample pad
[0117] The fiberglass film was cut into the size of 17 mm.times.4
mm and put into blocking solution A to be soaked, then taken out of
the solution and dried for 10 h at 39.degree. C. to obtain the
sample pad, and placed in a desiccator to store at room
temperature;
[0118] The blocking solution A included 1.5 g bovine serum albumin,
0.15 mL Triton X-100, 0.3 g polyvinylpyrrolidone, 4 g sucrose, 0.02
g sodium azide, 0.8 g sodium chloride, 0.29 g disodium hydrogen
phosphate dodecahydrate, 0.02 g potassium chloride, 0.02 g
potassium dihydrogen phosphate, to which water was added to reach
100 mL of final volume.
[0119] (4) preparation of the gold-labeled pad
[0120] The sample pad is cut into the size of 6 mm.times.4 mm, and
the nanogold-labeled anti-aflatoxin B.sub.1 monoclonal antibody
solution was transversely sprayed onto the sample pad by
spot-spraying, the amount of nanogold-labeled anti-aflatoxin
B.sub.1 monoclonal antibody required on per cm of spraying length
was 72 ng. After lyophilization under vacuum for 2 h, the
gold-labeled pad was prepared and placed in a desiccator to store
at room temperature;
[0121] The particle diameter of the nanogold in said nanogold
solution was 20 nm;
[0122] The method for preparation of nanogold-labeled
anti-aflatoxin B.sub.1 monoclonal antibody solution is described as
follows: 50.0 mL of commercially available nanogold solution was
taken with the concentration of 0.01%, and the pH of the solution
was adjusted to 5.5 with 0.1 mol L.sup.-1 potassium carbonate
aqueous solution; 2 mL of 0.1 mg mL.sup.-1 anti-aflatoxin B.sub.1
monoclonal antibody aqueous solution was added slowly with stirring
and the solution was further stirred for 30 min; 10% bovine serum
albumin aqueous solution was added until the final concentration of
bovine serum albumin is 1%, and the solution was further stirred
for 30 min; after standing at 4.degree. C. for 2 h, it was
centrifuged at 1500 rpm for 15 min, the supernatant was removed and
the pellet was discarded; the supernatant obtained was centrifuged
at 12000 rpm for 30 min, and the supernatant was discarded, then 50
mL label-washing preservation solution was added and the resulting
solution was centrifuged at 12000 rpm for 30 min again, the
supernatant was discarded, and the precipitate obtained was
resuspended with label-washing preservation solution to obtain the
concentrated solution with the volume of 5.0 mL, which was stored
at 4.degree. C. for use, wherein the mass concentration of the
nanogold-labeled anti-aflatoxin B.sub.1 monoclonal antibody
solution was 0.04 mg mL.sup.-1;
[0123] Said 0.1 mol L.sup.-1 potassium carbonate solution was
obtained as follows: 13.8 g potassium carbonate was dissolved in
purified water and diluted to 1000 mL of final volume, then
filtered by 0.22 .mu.m filter membrane; said 0.1 mg mL.sup.-1
anti-aflatoxin B.sub.1 monoclonal antibody solution was obtained as
follows: 1 mg of commercially available anti-aflatoxin B.sub.1
monoclonal antibody was diluted with water to 10 mL of final
volume; said 10% bovine serum albumin aqueous solution was obtained
as follows: 10 g bovine serum albumin was diluted with water to 100
mL, then filtered by 0.22 .mu.m filter membrane; said label-washing
preservation solution was obtained by mixing 2.0 g PEG-20000, 0.2 g
sodium azide and 0.1235 g boric acid, to which water was added to
1000 mL, followed by filtration through 0.22 .mu.m filter
membrane.
[0124] (5) assembly of the multi-test-line digital
immunochromatographic test strip for semi-quantitative detection of
aflatoxin B.sub.1
[0125] A water-absorbing pad, a detection pad, a gold-labeled pad
and a sample pad were adhered sequentially on one surface of said
paperboard from top to bottom, wherein each adjacent pads were
overlapped and connected, and the length of each overlapped part
was 2 mm, thus the multi-test-line digital immunochromatographic
test strip for semi-quantitative detection of aflatoxin B.sub.1 was
prepared (see FIG. 2).
[0126] The method for use of the multi-test-line digital
immunochromatographic test strip for semi-quantitative detection of
aflatoxin B.sub.1 is described as follows: the test samples of 1st
and 2nd which had been grinded finely were weighed, to which
methanol aqueous solution with the concentration of 60.about.80%
(v/v) was added, and the m/v ratio of the test sample and methanol
solution was 2 g mL.sup.-1. The solution was well-mixed and
extracted with ultrasound at 60.degree. C. water bath for 8 min,
then left for 8 min. The supernatant (i.e. the extract) was diluted
to 1:2.5 with water, resulting in that the final concentration of
methanol in the dilution was 24%. 100 .mu.L diluted sample solution
used as the test solution was added dropwise to the sample pad of
the multi-test-line digital immunochromatographic test strip for
semi-quantitative detection of aflatoxin B.sub.1. Said test strip
was used as the test strip for detection. In the meantime, 100
.mu.L of water was used as the negative control solution and added
dropwise to the sample pad of another multi-test-line digital
immunochromatographic test strip for semi-quantitative detection of
aflatoxin B.sub.1, which was used as the control test strip. The
results are read out after 15 min.
[0127] Result evaluation: the test lines of the test strip for
detection of the 1st test sample exhibited red lines, while the
control line did not exhibit a red line, thus the result of this
test strip for detection was considered as null, see FIG. 5-1: the
test lines of the test strip for detection of the 1st test sample
did not exhibit red lines, while the control line did not exhibit a
red line either, thus the result of this test strip for detection
was considered as null, see FIG. 5-2.
* * * * *